Disorders of the central nervous system are often associated with little or no recovery due to in part to poor neural regenerative capacity, damaging mechanisms that persist after initial neuronal injury, and inhibitory properties intrinsic to myelin. Regenerative medicine strategies that utilize synthetic or natural materials as permissive environments for maximizing axonal extension may not be sufficient for restoration of sight because axons that extend from each retina cross at the optic chiasm and selectively project to both hemispheres of the brain. Protein ligands, such as ephrin-B2, activate receptors on retinal ganglion cells to induce directional axon growth in order to form the divergent neural projections during embryogenesis. Development ligands such as these may be able to guide regenerating axons when engineered into biomaterials for tissue engineering. However, there is a critical need for physiologically- and translationally-relevant culture models that support the systematic investigation of structural and molecular parameters that may influence tissue growth. The overall goal of this project is to develop a 3D tissue culture model to study the guidance of retinal neurites in response to engineered cues that mimic the spatial distribution of ligands found at the optic chiasm during development. Specifically, we hypothesize that ephrin-B2, immobilized in a spatially-specific manner within a synthetic three-dimensional matrix, will selectively direct neurite outgrowth from embryonic retinal explants in a structural configuration that mimics the optic chiasm. In order to evaluate this hypothesis, we propose the following specific aims: Aim 1: Synthesize a photo-labile peptide hydrogel for localized immobilization of protein ligands. Aim 2: Develop a dual hydrogel platform for 3D retinal neurite outgrowth and incorporation of immobilized protein ligands. Aim 3: Evaluate the efficacy of ephrin-B2, locally immobilized in a 3D peptide hydrogel, to selectively direct neurite outgrowth from embryonic retinal explants. The techniques developed from this work will allow for the systematic manipulation of the spatial arrangement of structural and molecular cues for directing neuronal growth. Thus, it is anticipated that this work will establish a new experimental platform to study neural growth and guidance and also suggest potential treatment strategies to be explored in future studies. Disorders of the central nervous system, including optic neuropathies, are difficult to treat due to a poor capacity for nerves there to regenerate. In the optic nerve, even maximizing nerve axon regeneration may not lead to restoration of sight because nerves from each eye cross at the optic chiasm and project to specific regions at either side of the brain. The use of functional biomaterials that are able to selectively guide growing axons may suggest new treatment strategies for optic nerve and other central nervous system disorders.